CN101829103A - Application of flavonoid quercetin dimmer as medicament for treating viral hepatitis B - Google Patents
Application of flavonoid quercetin dimmer as medicament for treating viral hepatitis B Download PDFInfo
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Abstract
The invention relates to the application of flavonoid quercetin dimmer as the medicament for treating viral hepatitis B, in particular to the application of flavonoid quercetin dimmer or pharmaceutically acceptable salt thereof to the preparation of the medicament for eliminating HBsAg and HBeAg and inhibiting HBV DNA replication. The flavonoid quercetin dimmer or pharmaceutically acceptable salt thereof has obvious HBsAg and HBeAg inhibiting activity, and at the concentration of 100mcg/ml, the flavonoid quercetin dimmer pharmaceutically acceptable salt thereof has the HBsAg eliminating strength of 65.7% and the HBeAg eliminating strength of 44.8% which are respectively 4.1 times and 2.7 times higher than the positive control medicament of Alpha-interferon and has the HBV DNA inhibiting ratio of 44.8% which is 117% of the HBV DNA inhibiting ratio of the Alpha-interferon at the highest test concentration. Therefore, the flavonoid quercetin dimmer or pharmaceutically acceptable salt thereof can be expectedly used for preparing the non-nucleoside medicament for eliminating HBsAg and HBeAg, inhibiting HBV DNA replication and treating viral hepatitis B.
Description
Technical field
The present invention relates to medical technical field, particularly, the present invention relates to a kind of flavonoid quercetin dimmer or its officinal salt and be used to prepare the purposes that reduces hepatitis B virus surface antigen HBsAg and hepatitis B virus e antigen HBeAg, inhibition HBV dna replication dna, treats hepatitis b virus infected disease medicament.This dimer flavone has and suppresses HBsAg and HBeAg activity quite significantly, its intensity of removing HBsAg and HBeAg is respectively 65.7% and 44.8% under 100 mcg/ml concentration, surpasses 4.1 times and 2.7 times of positive control medicines (alpha-interferons of 10000 units per ml) respectively; Simultaneously, it demonstrates 44.8% suppression ratio to HBV DNA when this concentration, and the suppression ratio that is alpha-interferon when the highest test concentrations 117%.Above pharmacodynamic result shows that this flavonoid quercetin dimmer or its officinal salt can be expected and is used to prepare the purposes of removing HBsAg and HBeAg, inhibition HBV dna replication dna, the hepatitis b virus infected disease non-nucleoside medicine of treatment.
Background technology
Hepatitis B (hepatitis B) is the infectious disease that is caused by hepatitis b virus hbv.HBV is a member of Hepadnaviridae hepadnaviridae, and it is shaped as the spheroidal particle of diameter 42 nanometers.HBV is peculiar virus, and less being infectious in other animal has only in human body or primate chimpanzee body and just can duplicate.This virus is propagated by hepatitis B virus carriers and hepatitis B patients'blood, saliva, seminal fluid, vaginal secretions, has chronic carrier state.Hepatitis B is widely current in China, and because of it is divided in vertical transmission, horizontal transmission, the family propagation, iatrogenic infection and multiple mode such as spread through sex intercourse, to crowd infection rate's height, infection rate reaches more than 35% in certain areas.According to interrelated data, hepatitis detects male patient and has reached 1.89 hundred million, and the number (carrier) nearly 400,000,000 of should not going to a doctor is one of the most serious infectious disease of current harm people ' s health.Hepatitis B clinical manifestation variation easily develops into chronic hepatitis B (CHB) and liver cirrhosis, and a few patients can change primary hepatocarcinoma into.
Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and the HBsAg positive is a goldstandard of judging that HBV infects.The HBsAg positive but do not have the hepatitis symptom person of appearance and become HBV virus carrier.The HBsAg titre is big more, and it is just big more that it merges the active probability that raises of hepatitis B virus core antigen HBeAg, the HBV DNA positive and DNA polymerase, thereby infectiousness is strong more.In like manner, suppress the secretion of HBsAg and duplicate and also be an important target in the research and development anti-hepatic-B virus medicine and detect target.Report: hepatitis B HBsAg such as the refined cloud of the Wu of Beijing Ditan Hospital remove and there is certain dependency in hepatitis B closed loop covalency DNA (cccDNA), and removing HBsAg is the significantly reduced sign of cccDNA level.2002, deliver the researcher of result of study at " New England Journal of Medicine " and think: for CHB patient, remove as obtain HBsAg before liver cirrhosis, then its liver cirrhosis and hepatocarcinoma incidence rate will reduce by 60 times.All HBsAg serum is removed in the guide of the AASLD of U.S. hepatopathy research association, the Asia-Pacific liver APASL of research association and the EASL of EASL as one of treatment terminal point determining standard.
According to European liver EASD in 2008 annual meeting: Polyethylene Glycol Intederon Alpha-2a treatment CHB patient 48 week back drug withdrawals, followed up a case by regular visits to 1,2,3,4 year, its HBsAg clearance rate is respectively 3%, 6%, 8% and 11%, and 1,2,3,4 to be only be 0%, 0%, 0% and 3% after drug withdrawal to the HBsAg clearance rate with the lamivudine person separately.Thereby this interferon is considered to treat the negative CHB patient's optimal treatment selection of HBeAg.As seen find that the medicine that can efficiently remove HBsAg has important social and economic benefit.
Hepatitis B virus e antigen HBeAg is the structural protein of hepatitis B viruses (HBV) kernel, produces in a large number when hepatitis B virus is bred.Hepatitis B viruses (HBV) has in all known dna viruses minimum genome (only 3.2kb), its gene five kinds of albumen (S, C, E, P, X) of mainly encoding.C albumen is viral core protein, and the E albumen proteic part that is C becomes hepatitis B virus e antigen (HBeAg), and it is to have encoded but unassembled albumen in virion, can be secreted in the blood samples of patients when virus replication and go.Clinically, usually serum HBeAg is treated as hbv replication, infectiousness, the degree that is in a bad way and to it and reply the important symbol thing of estimating.This antigen and HBV DNA are closely related, are to express the very practical blood serum designated object of virus replication clinically.Blood serum designated object HBeAg positive patient illustrates in its body that hbv replication is arranged, so higher infectiousness is arranged.It is strong more that patient HBeAg expresses this patient's infectiousness of high more explanation.In like manner, suppress the secretion of HBeAg and duplicate and also be an important target in the research and development anti-hepatic-B virus medicine and detect target.HBeAg removes in the explanation body has lasting HBV to suppress, and ALT is normal, and the tissue inflammation necrosis alleviates, and the liver cirrhosis odds reduces.Therefore, serum HBeAg is believed to reflect more stable therapeutic effect, and HBeAg serum clear flag patient's immune system and begun to play a role.2002, deliver the researcher of result of study at " New England Journal of Medicine " and think: for CHB patient, remove as obtain HBeAg before liver cirrhosis, then its liver cirrhosis and hepatocarcinoma incidence rate will reduce by 10 times.All HBeAg serum is removed in the guide of the AASLD of U.S. hepatopathy research association, the Asia-Pacific liver APASL of research association and the EASL of EASL as one of treatment terminal point determining standard.So, can suppress, reduce HBeAg expression or active medicine and also promptly belong to treatment hepatitis B virus infection active drug.
At present, the medication to hepatitis B patient mainly is divided into several big classes such as the liver protecting and ALT lowering, antiviral, anti-hepatic fibrosis and adjusting immunity.Antiviral is a basic method, and the liver protecting and ALT lowering is an auxiliary treatment, takes stopgap measures and rarely seen effecting a permanent cure more.The research that still is the antiviral treatment aspect that made progress in the last few years.Yet, can only reach for viral hepatitis B therapeutic scheme clinically at present and suppress hbv replication and secondary infection, main medicine is still nucleoside medicine such as lamivudine (3-TC), Entecavir, adefovirdipivoxil (ADV), Sebivo etc., is in the interim emtricitabine of clinical trial, tenofovir, clevuding etc. in addition.In China, lamivudine has become the medical insurance medication, is applied to large quantities of HBV patients.Nucleoside medicine part advantage is: the bioavailability height, and oral safer.Yet, though their disease controlling, fetch long prices first effectively; Second life-time service all drug resistance can occur, and the bounce-back in various degree that occurs HBV DNA, ALT and liver histological after the drug withdrawal; The 3rd, the comparatively tangible well-known ill effect that the life-time service nucleoside medicine occurs, for example kidney injury, baby's teratogenesis etc.Headache is the most: virus is drug-fast cure rate to occur greatly reducing, because nucleoside medicine is reversible to virus replication, thus to most of patient if desire to reach greatest treatment efficacy, the course of treatment must be more than 1 year, so its drug resistance occurs thereupon, does not just reach the effect of expection.Nucleoside medicine be difficult in addition remove cccDNA, treatment after 1 year HBsAg be difficult to weak points such as cloudy commentaries on classics.
The biological engineering class antiviral drugs that interferon (α, beta-interferon) and recombinant interferon class etc. derive from human leukocyte becomes research and treatment CHB focus medicine in the recent period, and it has antiviral and immunomodulating dual function.Thereby it both can suppress virus replication by antivirus action and alleviate the liver cell inflammatory reaction, reduces hepatocellular damage, plays and improves patients clinical symptom and liver physiological function thereby delay PD; Again can the enhance immunity effect, by the effect of natural killer cell in the intensive aspect and helper T lymphocyte, especially can promote killer T cell to go to kill and wound by virus infected cell, therefore play antivirus action indirectly.The HBsAg serology conversion that interferon therapy CHB patient obtains is more more lasting than lamivudine, and " digestive tract " magazine the next item up studies show that in 2003: 3 years relapse rates of interferon therapy group HBsAg significantly are lower than the lamivudine group; And long-acting interferon can use weekly once, and is comparatively convenient.Therefore, interferon day by day becomes one of choice drug that is used for the treatment of chronic viral hepatitis B virus clinically, but its side effect and untoward reaction report are more, total effective rate is not high, cost an arm and a leg, patient economy burden is big, thereby causes and be difficult to clinically be extensive use of, and the decompensated cirrhosis patient is not suitable for using.
Along with the research of hepatopathy, developed the analysis of standardized HBV DNA in recent years, advanced understanding greatly the hepatitis B patient state of an illness.The quantitative analysis of HBV DNA can be predicted the seriousness and the prognosis thereof of hepatitis B, because HBV DNA lasting masculin (promptly continuing viremia) makes the hepatitis B disease progression easily and increases the weight of; High hepatitis B virus (HBV DNA) content promotes the formation of liver cirrhosis easily; The lasting existence of HBV DNA is that the high risk factor, particularly viral level of hepatocarcinoma (HCC) generation is higher, the course of disease is long, older or merge other liver patient; The HBV DNA that continues high concentration exists, and the mortality rate that can cause losing compensatory liver cirrhosis and constitutional liver serious symptom obviously increases.Simultaneously must recognize that the relation of HBV dna level and liver histological is extremely close: bibliographical information is through antiviral therapy, and the improvement of hepatic fibrosis and elimination are obviously; Recent international hepatopathy meeting report, potent and low chemical sproof antiviral therapy along with the reduction of HBV DNA with turn out cloudy, in various degree reverse can occur and observe liver cirrhosis, and therefore present opinion liver cirrhosis also should carry out antiviral therapy.
Therefore, the application of HBV DNA index in antiviral therapy also plays a part very important: the level of HBV DNA is the important indicator whether the decision chronic hepatitis B needs antiviral therapy; Make the difference treatment standard and the requirement of the HBeAg positive or HBeAg feminine gender respectively according to the different situations of HBV DNA; In antiviral therapy, according to the therapeutic response of HBVDNA, judge whether that virusology replys in early days and then determine the strategy of long-term prescription to reply with the virusology that obtains persistence, reach and continue the purpose that virus suppresses; According to the virological situation of replying of HBVDNA, create serological switching foundation of HBeAg and condition, to reach its good middle therapeutic goal; Continue the inhibition situation according to HBV DNA and strive for that virus continues feminine gender, to strive for reaching the final therapeutic goal of antiviral; Continue to be suppressed fully according to HBV DNA, also demonstrated improvement in various degree and the disappearance of cccDNA; In antiviral therapy, assess and prevent the risk of caused virus variation of antiviral drugs and drug resistance generation with the variation of HBV DNA; In case when virus variation or drug resistance took place, the variation of HBV DNA was unique signal at first and diagnosis basis, also be treatment drug resistance and guidance and the foundation that changes therapeutic strategy.In sum, the inhibition degree of HBV DNA there is new important meaning in the further diagnosis of hepatitis B and treatment,, assessment hepatitis B prognosis and drug resistance danger is all had bigger directive function the observation of curative effect.
By above-mentioned factor as can be known: the basic link of another one that the inhibition hepatitis B virus is bred in vivo is to suppress duplicating of HBV DNA.The reduction of HBV dna level or be lower than detection level be the check antiviral drugs other one JINYAOSHI.So, Asia-Pacific liver EASD and European liver EASD all with the HBV DNA detection less than as one of hepatitis B virus patient treatment terminal point.Also tested compounds is considered as one of the test event that must finish for the inhibition strength of HBV DNA in China's new drug development guide.Why lamivudine can become first-selected nucleoside medicine is because it has the activity of potent inhibition HBV dna replication dna.Therefore can suppress the novelty medicine that chemical compound that the HBV dna replication dna can suppress HBsAg again will more promise to be the treatment hepatitis B patient.
Mandatory declaration be: the present antiviral drugs of the using inhibitor of virus replication just in fact, directly kill virus and break virus body, otherwise will damage host cell.These antiviral drugs (mostly being nucleoside medicine) also exist above-mentioned toxic and side effects greatly, easily to cause after viral gene sudden change, the drug withdrawal shortcomings such as easily knock-on, so the development of new antiviral drugs is the task of top priority in current medicament research and development field.It all has extremely important social meaning and economic implications for a large amount of hepatitis B patient and virus carrier, the control sources of infection etc. of treatment China.So, new non-nucleoside hepatitis B virus inhibitor and this type of lead compound that can reduce HBsAg, HBeAg or inhibition HBV dna replication dna of discovery has very big guiding significance from the natural drug of national folk life-time service, and vast development prospect is arranged.
Based on this purpose, the inventor once finished the patent and the article of multinomial anti-hepatitis virus natural product and structure of modification derivant thereof in the past, multiple inhibition hepatitis B virus surface antigen HBsAg or hepatitis B virus e antigen HBeAg activity have been found, the chemical compound that suppresses the HBV dna replication dna, thus illustrating to filter out from natural product and synthesis of derivatives thereof to reduce HBsAg or HBeAg, the novelty medicine of control hepatitis B virus infection is feasible [referring to " medical usage of a class mapping eucalyptus globulus alkanol type sesquiterpene for inhibiting hepatitis virus " (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo etc.; ZL 200610053827.4); " medical usages of 2 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Lee school Kun, Zhao Yu, Huang Kexin, Li Haibo etc.; ZL200610053749.8); " 2 α, the medical usage of 3 beta-dihydroxies-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Zhang Li and, Dong Sun Han, Li Haibo etc.; ZL200610053601.4); The purposes and the pharmaceutical composition thereof of hepatitis B virus " the eremophilane lactone suppress " (Zhao Yu, Li Haibo, Yang Leixiang, girth are new etc.; ZL 03153691.3); " a kind of Eremophilone lactones acid natural product and application thereof " (Zhao Yu, girth new, Shi Shuyun, Wang Xiaoyu etc.; ZL 200610053575.5); " a kind of eudesmane type sesquiterpenes acid and uses thereof " (Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei etc.; ZL 200610053579.3); " purposes of laggera plant abstract in inhibiting herpes simplex virus and hepatitis B virus " (Zhao Yu, girth new, Yu Rongmin, Bai Hua; CN 1989989A); " medical usage of 1 β-oxo-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Li Haibo etc.; CN 1927197A); " medical usage of 1 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Wu Xiumei etc.; CN 1935131A); " medical usage of acid of mapping eremophilane and inhibition hepatitis B surface antigen thereof " (Huang Kexin, Lee school Kun, Wang Xiaoyu, Zhao Yu etc.; CN 101239054); " 1-oxygen-substituted benzene formyl quinic acid chemical compound and inhibition hepatitis B virus purposes thereof " (Lee school Kun, Hu Lihong, Wu Xiumei, Zhao Yu etc.; CN101293836); Inhibition HBsAg, HBeAg that the inventor has delivered and HBV DNA isoreactivity article are referring to " In Vitro Antiviral Activity of Three EnantiomericSesquiterpene Lactones from Senecio Species Against Hepatitis B Virus ", Haibo Li (Li Haibo), Changxin Zhou (girth is new), Xiumei Wu (Wu Xiumei), Yu Zhao* (Zhao Yu) etc., Antiviral Chemistry﹠amp; Chemotherapy, 2005,16,277-282; " Evaluation of Antiviral Activity of Compounds Isolated fiomRanunculus sieboldii Miq.and Ranunculus sceleratus L ", Haibo Li (Li Haibo), Changxin Zhou (girth is new), Xiumei Wu (Wu Xiumei), Yu Zhao* (Zhao Yu) etc., Planta Medica, 2005,71 (12), 1128-1133; " Application ofhigh-speed counter-current chromatography for the isolation of antiviraleremophilenolides from Ligularia atroviolacea ", Shi, Shu-Yun (Shi Shuyun), Hai-Bo (Li Haibo), Zhao, Yu (Zhao Yu) etc., Biomedical Chromatography, 2008,22 (9), 985-991; " Purification and identification of antiviralcomponents from Laggera pterodonta by high-speed counter-currentchromatography ", Shuyun Shi (Shi Shuyun), Yu Zhao (Zhao Yu) etc., Journalof Chromatography B, 2007,859,119-124].Undoubtedly, continuation is sought from natural product and structure of modification derivant thereof, and can to remove lead compound that HBsAg or HBeAg, inhibition HBVDNA duplicate be unusual being necessary property and urgent, also therefore classified as one of great special project of new drug development by the Ministry of Science and Technology.
Quercetin is a kind of flavonol, and discovery Quercetins such as Hirose can form dimer [Hirose etc., Chemistry Letters, 1999,8,775-776].Because it than greatly almost one times of Quercetin molecular weight, contains more polyhydroxy isoreactivity group in the molecule, it perhaps has the anti-virus ability that is different from Quercetin.(left side is state-run for the state-run grade in a left side, Liu Shuling, Xu Guili, world Chinese digests magazine, 2006 14 13 phases of volume, the 1241-1246 page or leaf) summarized the active progress of the external anti-HBV of medicinal plants composition over nearly 20 years, the multiple natural product of touching upon in the literary composition, do not report that wherein any flavonoid quercetin dimmer chemical compound has the active relative recording of anti-HBV, only it has been carried out preparation and the anti-HBV activity research that forefathers do not add the novel synthesis of attention by inventor team.One of our purpose is: wish to find to reduce the dimer flavone lead compound of HBsAg or HBeAg, inhibition HBV dna replication dna, have the novelty medicine that can remove HBsAg or HBeAg, inhibition HBV dna replication dna, treatment CHB thereby it is developed further into.Finish the present invention in view of the above.
Summary of the invention
The purpose of this invention is to provide the flavonoid quercetin dimmer shown in the formula (1) or its officinal salt and be used for the new purposes that preparation is removed HBsAg or HBeAg, inhibition HBV dna replication dna, treated the hepatitis B medicine.
The name of formula (1) chemical compound is called: 1,3, and 11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone-2)-5a-(3, the 4-dihydroxy phenyl)-5,6,11-six hydrogen-5,6,11-three oxygen-aphthacene-12-ketone.
The present invention also provides the method for the flavonoid quercetin dimmer compounds shown in a kind of preparation formula (1), it is characterized in that:, have silver catalyst to carry out free radical coupling reaction coupling and get under anhydrous condition with commercially available or homemade Quercetin.
Another object of the present invention has provided a kind of pharmaceutical composition of removing HBsAg or HBeAg, inhibition HBV dna replication dna, treatment hepatitis B that is used to prepare, and it is characterized by by containing the mixture that (1) chemical compound of the formula as active component for the treatment of effective dose or its officinal salt and pharmaceutically acceptable auxiliaries are formed.Its pharmaceutical dosage form can be that tablet, capsule, injection, aerosol, suppository, membrane, drop pill, paster agent, subcutaneous planting bury agent, externally-applied liniment, oral liquid or ointment, can also adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.
Flavonoid quercetin dimmer chemical compound of the present invention (1) is compared with natural flavone Lignanoids compounds Quercetin, feature with differentiation on many structures and the physico-chemical property comprises that speciality such as its hydrophobicity, armaticity, Gibbs free energy, hydrogen bond receptor, electrical, intermolecular Van der Waals force and 3D conformation, direction of extension, molecule center of gravity, conjugated degree, electrical distribution center all have obviously different with Quercetin; And chemical compound (1) molecular weight ratio Quercetin has increased 300 mass units.Above-mentioned feature has all determined the three-dimensional conformation of chemical compound shown in the formula (1) all may produce bigger difference with the ligand-receptor that the 3d space structure of HBsAg or HBeAg and even HBV DNA combines in conjunction with complex form and combination, its binding site and binding pattern, it all can produce bigger change in conjunction with free energy etc., thereby may beyond thought effect be arranged aspect HBsAg or HBeAg, the inhibition HBV dna replication dna removing.
We have tested the growth inhibited effect of this chemical compound to the HepG2.2.15 cell, have tested it simultaneously to B-mode HBsAg, the HBeAg of HepG2.2.15 emiocytosis and the inhibition activity that HBVDNA is duplicated.Result of the test is found: the synthetic flavonoid quercetin dimmer that obtains has the extremely significant activity that suppresses to the HBsAg and the HBeAg of HepG2.2.15 emiocytosis among the present invention, its intensity of removing HBsAg and HBeAg is respectively 65.7% and 44.8% under 100 mcg/ml concentration, surpasses 4.1 times and 2.7 times of positive control medicines (alpha-interferons of 10000 units per ml) respectively; Simultaneously, it demonstrates 44.8% suppression ratio to HBV DNA when this concentration, and the suppression ratio that is alpha-interferon when the highest test concentrations 117%.More than all formula (1) chemical compound beyond thought anti-HBV effect is arranged, thereby can expect that it can be used as the active lead compound of removing HBsAg or HBeAg, inhibition HBV dna replication dna, treatment hepatitis B and continues exploitation.
Through the detailed Literature Consult of the inventor, up to the present, still there is not report about this compounds for treating hepatitis B virus infection disease and preparation anti-hepatic-B virus medicine.Formula (1) chemical compound flavonoid quercetin dimmer all belongs to beyond thought discovery for HBsAg, HBeAg and the potent inhibition of HBV DNA, and definite originality is arranged.In sum, uniqueness on our synthetic this flavonoid quercetin dimmer existing structure, the novelty that the research of antivirus action aspect is arranged again, and in anti-hepatitis B activity test, both found the activity of very potent inhibition HBsAg, found the activity of significant inhibition HBeAg again, found that also it has the inhibition HBV dna replication dna activity of extremely strong effect; The lead compound that is expected to become potent removing HBsAg or HBeAg, inhibition HBV dna replication dna and treats the non-nucleoside medicine of CHB.
Usefulness of the present invention is: the flavonoid quercetin dimmer shown in the discoverable type (1) has the effect of removing HBsAg or HBeAg, inhibition HBV dna replication dna first, with and and in the patent medicine potentiality aspect the control hepatitis B virus infection disease, the non-nucleoside original new drug that remove hbs antigen HBsAg or hepatitis B virus e antigen HBeAg for exploitation becomes treatment hepatitis B virus original new drug, exploitation, suppresses the HBV dna replication dna provides new material base.Have potential huge social benefit and economic benefit.The present invention's characteristics again is: the present invention's synthetic starting material convenient sources, and synthetic convenient.Its preparation method is simple, and raw material sources conveniently are easy to get, and cost is low, pollutes for a short time, is beneficial to the large-scale production under the energy-saving and emission-reduction condition.Industrialization prospect is very clear and definite.
The specific embodiment
The inventor is by chemosynthesis, and by multiple chromatography means purification obtain this can potent inhibition hepatitis B HBsAg and the secretion of HBeAg can effectively suppress the active flavonoid quercetin dimmer class reactive compound of HBV dna replication dna again, derive its chemical constitution through integration analysis such as mass spectrum and NMR (Nuclear Magnetic Resonance) spectrum again.The inventor finds, formula (1) chemical compound does not have obvious inhibitory action to the growth of HepG2.2.15 cell, and the hepatitis B HBsAg of HepG2.2.15 emiocytosis and secretion and the duplicating of HBV DNA of HBeAg are had significant inhibitory effect, point out this chemical compound to have drug safety, potent removing HBsAg or HBeAg, reach and suppress HBV dna replication dna characteristics of high efficiency.Therefore, according to the inventor's research, the flavonoid quercetin dimmer chemical compound shown in the designed and synthetic formula of inventor (1) can be used to prepare the non-nucleoside medicine of treatment hepatitis B virus infection disease and be used for the treatment of the hepatitis B virus infection disease.
In order to understand essence of the present invention better, use the preparation of formula (1) chemical compound below respectively and, its new purposes in pharmaceutical field is described to the HepG2.2.15 cell growth inhibition and to the result of the inhibitory action test of HBsAg, the HBeAg of HepG2.2.15 emiocytosis and HBV dna replication dna.Embodiment has provided partial synthesis, the structure of formula (1) chemical compound and has identified and activity data.Mandatory declaration, embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1:The preparation of formula (1) chemical compound
1.1 instrument and reagent:
Ultraviolet spectra is measured with Shimadzu UV-240 ultraviolet spectrophotometer; Proton nmr spectra
1H-NMR measures (tetramethylsilane ether TMS is interior mark) by INOVA type NMR spectrometer with superconducting magnet (VARIAN INOVA-400MHz); Electrospray Mass Spectrometry ESI-MS is measured by BrukerEsquire 3000+ mass spectrograph, and column chromatography is produced by Haiyang Chemical Plant, Qingdao with silica GF254 (10-40 order) with silica gel (100-200,200-300 and 300-400 order) and thin layer chromatography; Agents useful for same is analytical pure, thin layer preparative chromatography (PTLC) the aluminium foil silica gel plate of Merck company; Column chromatography adopts Sweden Amersham Pharmacia Biotech AB company product with polydextran gel Sephadex LH-20; Reverse phase silica gel RP-18 adopts the Chromatorex product of Japanese Fuji Silysia Chemical company; MCI is a Mitsubishi chemical company product, and thin plate (TLC) detects the uviol lamp with 254nm and 365nm; Developer iodine vapor, 10% sulphuric acid-ethanol and phosphorus molybdenum acid solution.
1.2 the preparation of formula (1) chemical compound:
Add 343 milligrams of Quercetins (this chamber prepares voluntarily, and HPLC detects purity 98%) in exsiccant reaction bulb, 550 milligrams of Disilver carbonates add 20 milliliters of dry acetone and 60 milliliters of anhydrous benzene solution of crossing down in nitrogen protection, stir 20 hours between 55-60 ℃.Mixture leaves standstill, the filtering insoluble matter.The mother solution concentrating under reduced pressure gets yellow solid.With 200～300 order silica gel column chromatographies, with chloroform/methanol (20: 1) eluting.Get 182 milligrams of yellow solids.R
f(chloroform: methanol=10: 1)=0.18; Infrared IR (KBr tabletting) cm
-1: 3195,1689,1647,1588,1495; Proton nmr spectra
1H NMR (400MHz, deuterated acetone): 6.05 (bimodal, J=2.0Hz, 1H, H-6 '), (6.10 bimodal, J=2.0Hz, 1H, H-8 '), 6.28 is (bimodal, J=2.0Hz, 1H, H-6), 6.62 is (bimodal, J=2.0Hz, 1H, H-8), 6.81 is (bimodal, J=8.4Hz, 1H, H-13), 7.16 (double doublets, J=2.4,8.4Hz, 1H, H-14 '), (7.29 bimodal, J=8.0Hz, 1H, H-13 '), (7.35 bimodal, J=2.4Hz, H-10 '), 7.96 is (bimodal, J=2.0Hz, 1H, H-10), 8.00 (double doublets, J=2.0,8.8Hz, 1H, H-14); Carbon-13 nmr spectra
13C NMR (100MHz, deuterated acetone, δ ppm): 91.4 (C-3 '), 94.6 (C-8), 97.3 (C-8 '), 98.0 (C-6 '), 99.3 (C-6), 100.9 (C-4 ' a), 101.6 (C-2 '), 104.2 (C-4a), 115.4 (C-9 '), 116.5 (C-14 '), 117.7 (C-10), 118.2 (C-13), 121.1 (C-10 '), 123.4 (C-14), 126.2 (C-9 '), 126.9 (C-13), 137.6 (C-3), 141.8 (C-11), 143.0 (C-12), (145.4 C-2, C-11 '), 147.6 (C-12 '), 157.8 (C-8a), 160.7 (C-8 ' a), 162.3 (C-4a), 165.1 (C-5 '), 165.2 (C-7), (169.2 C-7 '), 176.7 (C-4), 189.0 (C-4 '); Electrospray Mass Spectrometry ESI-MS:m/z 603[M+H]
+(100).
Embodiment 2:Chemical compound (1) is to the inhibitory action of the hbs antigen (HBsAg) of HepG2.2.15 emiocytosis
2.1 cell culture:
In containing 10% inactivated fetal bovine serum, 100U/ ml penicillin and 100U/ milliliter streptomycin in the DMEM culture medium of 100 mcg/ml G418, are put 37 ℃, 5%CO with the HepG2.2.15 cell culture
2, cultivate in the incubator of 100% relative humidity.
Measure the inhibitory action of formula (1) chemical compound 2.2 adopt mtt assay to the growth of HepG2.2.15 cell:
The take the logarithm HepG2.2.15 cell of trophophase becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2, cultivate the chemical compound (1) that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 1000,200,40 and 8 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, cultivate after 72 hours, every hole adds MTT reagent 10 microlitres, continues to cultivate 4 hours, discards culture medium, every hole adds DMSO 200 microlitres, with agitator vibration 20 minutes, measures the OD value with microplate reader under the 570nm wavelength.With the culture hole that only adds culture medium is control wells.Suppression ratio (%)=control wells OD value-experimental group OD value)/control wells OD value * 100%.
Measure the inhibitory action of 1 pair of hbs antigen of chemical compound (HBsAg): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5/ milliliter is inoculated in 96 porocyte culture plates, 100 milliliters in every hole, and at 37 ℃, 5%CO
2, cultivate the sample that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 100,20 and 4 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.With HBsAg concentration in the ELISA kit measurement culture medium, represent with P/N; With the positive contrast 1 of lamivudine (3-TC) (test concentrations is 100,20 and 4 mcg/ml); With the positive contrast 2 of alpha-interferon (test concentrations is 10000,5000 and 1000 units per ml).The experiment triplicate.
2.3 experimental result:
Experimental result is as shown in table 1, and formula (1) chemical compound has the effect of significant inhibition hbs antigen (HBsAg).Its growth to the HepG2.2.15 cell does not have obvious inhibitory action, is higher than lamivudine and alpha-interferon but the HBsAg of HepG2.2.15 emiocytosis was suppressed activity under the high dose the 8th day the time.
Table 1. sample in the time of the 8th day to the excretory hbs antigen suppression ratio of HepG2.2.15
2.4 presentation of results:
This embodiment presentation of results: the flavonoid quercetin dimmer chemical compound shown in the formula (1) has significant inhibitory effect to the hbs antigen (HBsAg) of HepG2.2.15 emiocytosis, its intensity of removing HBsAg is 65.7% under 100 mcg/ml concentration, surpasses 4.1 times of positive control medicines (alpha-interferons of 10000 units per ml); Also surpass 4.1 times of positive control 2 lamivudines; As seen this flavonoid quercetin dimmer has very strong inhibition viral secretory surface antigen activity.
It is the state of approaching healing clinically that HBsAg removes, and for hepatitis B patient, its HBsAg removes becomes very valuable CHB treatment terminal point.Thereby the flavonoid quercetin dimmer chemical compound shown in the formula (1) can be expected and developed into the non-nucleoside original new drug that reduces hbs antigen, control Type B viral hepatitis symptom.
Embodiment 3:Chemical compound (1) is to the inhibitory action of the hepatitis B e antigen (HBeAg) of HepG2.2.15 emiocytosis
3.1 cell culture: method is with embodiment 2.
3.2 adopting mtt assay measures by the inhibitory action of formula (1) chemical compound to the growth of HepG2.2.15 cell: method is with embodiment 2.
3.3 measure the inhibitory action of chemical compound to hepatitis B e antigen (HBeAg): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5/ milliliter is inoculated in 96 porocyte culture plates, 100 milliliters in every hole, and at 37 ℃, 5%CO
2, cultivate the sample that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 100 mcg/ml, 20 mcg/ml and 4 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.With HBeAg concentration in the ELISA kit measurement culture medium, represent with P/N; With the positive contrast 1 of lamivudine (3-TC) (annotate: the lamivudine test concentrations is 100 mcg/ml, 20 mcg/ml and 4 mcg/ml); With the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).
3.4 experimental result: experimental result is as shown in table 2, and formula (1) compound exhibits goes out the effect of the inhibition hepatitis B e antigen (HBeAg) of highly significant.It does not have obvious inhibitory action to the growth of HepG2.2.15 cell under 40 mcg/ml concentration, but its intensity of removing HBeAg is 44.8% under 100 mcg/ml concentration, surpasses 2.7 times of positive control medicines 2 (alpha-interferons of 10000 units per ml); And positive control 1 lamivudine when maximum concentration (100 mcg/ml) HBeAg to be suppressed activity be zero.
Table 2. sample in the time of the 8th day to the excretory hepatitis B e antigen suppression ratio of HepG2.2.15
3.5 presentation of results: it does not have obvious inhibitory action to the growth of HepG2.2.15 cell the flavonoid quercetin dimmer chemical compound shown in the formula (1) under 40 mcg/ml concentration, but it has the inhibition HBeAg activity of highly significant, but its intensity of removing HBeAg is 44.8% under 100 mcg/ml concentration, surpasses 2.7 times of positive control medicines 2 (alpha-interferons of 10000 units per ml); More surpass and show the active positive control lamivudine of inhibition.As seen this flavonoid quercetin dimmer has significant inhibition hepatitis B virus secretion HBeAg activity, thereby can expect and develop into the medicine that reduces hepatitis B e antigen, control Type B viral hepatitis symptom.
Embodiment 4:The inhibitory action that formula (1) chemical compound duplicates the hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA) of HepG2.2.15 emiocytosis
4.1 cell culture: method is with embodiment 2.
Measure the inhibitory action of the flavanolignan's chemical compound shown in the formula (1) to the growth of HepG2.2.15 cell 4.2 adopt mtt assay: method is with embodiment 2.
4.3 the inhibitory action that the flavonoid quercetin dimmer chemical compound shown in the mensuration formula (1) duplicates hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2Cultivate the flavonoid quercetin dimmer chemical compound that adds after 24 hours with shown in the formula (1) of culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 100 mcg/ml, 20 mcg/ml and 4 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.Measure HBV DNA concentration in the culture medium with HBV DNA quantitative PCR kit in the time of the 8th day.(annotate: the lamivudine test concentrations is 100 mcg/ml with the positive contrast 1 of lamivudine (3-TC), 20 mcg/ml and 4 mcg/ml), with the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).
4.4 experimental result: experimental result illustrates as shown in table 3, and flavonoid quercetin dimmer formula (1) chemical compound has the effect of potent inhibition hepatitis B virus DNA replication.
Table 3 sample in the time of the 8th day to the suppression ratio of the HBV dna replication dna of HepG2.2.15 cell
4.5 presentation of results: the flavonoid quercetin dimmer chemical compound shown in the formula (1) has very potent inhibitory action to duplicating of hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA), it demonstrates 44.8% suppression ratio to HBV DNA under 100 mcg/ml concentration, and the suppression ratio that is alpha-interferon when the highest test concentrations 117%.Therefore, this flavonoid quercetin dimmer chemical compound belongs to significantly effectively non-nucleoside inhibition hepatitis B virus natural product, is worth very much further paying close attention to and further investigation, and can expects that further optimized development is to suppress the non-nucleoside original new drug that HBVDNA duplicates.
When above-mentioned description elaboration was of the present invention, the purpose that embodiment is provided simultaneously was to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of in entering claim of the present invention and its equivalent scope, practical application of the present invention comprises all general variations, cooperates, or improves.
Claims (4)
1. have the flavonoid quercetin dimmer of structure shown in the formula (1) or the purposes that its officinal salt is used for preparation treatment hepatitis B medicine;
2. the flavonoid quercetin dimmer or its officinal salt that have structure shown in the formula (1) are used for the purposes that preparation reduces hepatitis B virus surface antigen HBsAg medicine,
3. the flavonoid quercetin dimmer or its officinal salt that have structure shown in the formula (1) are used for the purposes that hepatitis B virus e antigen HBeAg medicine is removed in preparation,
4. the flavonoid quercetin dimmer or its officinal salt that have structure shown in the formula (1) are used for the purposes that preparation suppresses hepatitis B virus DNA (deoxyribonucleic acid) HBV dna replication dna medicine,
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| CN102000059A (en) * | 2010-10-27 | 2011-04-06 | 大理学院 | Medicinal application of querecetin dipolymer flavonoids to preparation of glycosidase enzyme inhibitors |
| CN104804014A (en) * | 2015-03-27 | 2015-07-29 | 云南民族大学 | Icetexane type diterpene dimer compound as well as preparation method and application thereof |
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| 《中医药学刊》 20050731 张尚斌等 "乙肝解毒胶囊的工艺研究" 第1322-1323页 1-4 第23卷, 第7期 2 * |
| 《中国中医药信息杂志》 20080531 辛秀等 "槲皮素对肝脏的药理作用研究进展" 第102-104页 1-4 第15卷, 2 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102000059A (en) * | 2010-10-27 | 2011-04-06 | 大理学院 | Medicinal application of querecetin dipolymer flavonoids to preparation of glycosidase enzyme inhibitors |
| CN102000059B (en) * | 2010-10-27 | 2012-08-22 | 大理学院 | Medicinal application of querecetin dipolymer flavonoids to preparation of glycosidase enzyme inhibitors |
| CN104804014A (en) * | 2015-03-27 | 2015-07-29 | 云南民族大学 | Icetexane type diterpene dimer compound as well as preparation method and application thereof |
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